BL Solution: 1 pack (3 g) of Barley leaf (Trade name: Oomugi Wakaba Funmatu 100%, Yamamoto Kanpou Seiyaku Co.) is added to 1,000 ml distilled water and boiled for 10 minutes, while stirring. The solution is passed through the qualitative filter paper.
EY Solution: 9 g of crushed yolk of hard-boiled egg is added to 1,000 ml distilled water and boiled for 10 minutes, while stirring. The solution is passed through the qualitative filter paper.
BL and EY Solution are mixed. The mixed solution is dilution with distilled water up to 6,000 ml, and then sterilized by autoclaving.
L Solution: Parts of light green leaves of lettuce is dried at 90 degrees C for about 16-18 hr without scoring. 3 g of the dried leaves is added to 1,000 ml boiling water (9:1 distilled water/tap water) and boiled for 30 minutes, while stirring. The supernatant is passed through sanitary cotton layers.
E Solution: 3 g of crushed yolk of hard-boiled egg is added to 1,000 ml water (9:1 distilled water/tap water) and boiled for 30 minutes, while stirring. The supernatant is passed through cotton layers.
Equal quantities of L and E Solution are mixed. pH of the solution is adjusted to 6.8 with 1N NaOH or 1N HCl. Next the solution is sterilized by autoclaving.
We use activated sludge of municipal treatment plant indicating the degree of treatment efficiency. MLSS of the sludge is adjusted about 5,000 mg/L, then autoclaved (121 degrees C, 30 minutes). The supernatant is passed through cotton batting. Next, pH of the solution is adjusted to 6.8-7.0 with 1N NaOH or HCl. The solution is sterilized by autoclaving.
A Solution: Glucose 0.15 g, Na-acetate 0.15 g, (NH4)2SO4 0.05 g, MgSO4/7H2O 0.025 g, KCl 0.025 g, CaCO3 0.025 g, Agar-extract 100 mL (20 g of agar powders is added to 1,000 mL distilled water, and then keep still overnight in room temperature. The supernatant is filtered by GFC.), distilled water 900 mL.
B Solution: 0.2 M Phosphate buffer 5 ml/L. A-liquid 61 mL and B-liquid 39 mL mixed and then autoclaved.
A-liquid: Na2HPO4/12H2O 71.64 g/L
B-liquid: NaH2PO4/2H2O 31.21 g/L
C Solution: FeCl3/6H2O 0.0005 g/L.
D solution: Vitamin B12 5x10-6 g/L, Vitamin B1 1x10-4 g/L (Keep the stock solution keep in a deep freezer. Before use, sterilize by filtration through a milipore filter (pore diameter: 0.22 um).
E Solution: Stigmasterol 0.01 g/L. Stigmasterol 50 mg is soluble 10 mL ethanol (99.99%).
A Solution is sterilized by autoclaving, then B, C, D, E solution are added.
For bacteria-feeders: These bacteria are mixed for food.
Micrococcus luteus (IAM 1313)
Acinetobacter calcoaceticus (IAM 1517)
Flavobacterium lutescens (IAM 1667)
Pseudomonas ovalis (IAM 1002)
Bacillus cereus (IAM 1029)
Klebsiella pneumonaie (IAM 1102)
Escherichia coli (IAM 1239)
Bacillus sabtilis (IAM 1069)
Stock culture: Standard agar medium, slant, 10 degrees C.
Liquid culture: LE medium, Tube, 20-30 degrees C.
For filamentous bacteria-feeders:
Sphaerotilus natans (SY-1) (Kohno strain)
Stock culture: Slant, 20 degrees C, 1/2CG medium (Bact-Casiton 0.25 g/L, Glucose 0.5 g/L, Yeast extract 0.25 g/L, tap water keeping one overnight in room temperature 1 L).
Mass culture: 30 degrees C, 300 mL flask (liquid volume 100 mL), culture period 1 day, centrifugation condition (10,000 rpm, 5 minutes), washing solution (1/750 M phosphate buffer).
Type 021N (T2-1, SNR-3) (Kohno strain)
Stock culture: Slant, 25 degrees C, EGG medium (Glucose 0.3 g/L, Na-acetate 0.3 g/L, (NH4)2SO4 0.1 g/L, MgSO4/7H2O 0.05 g/L, KCl 0.05 g/L, CaCO3 0.05 g/L, Agar 10 g/L, distilled water 1 L, 0.2M phosphate buffer solution 5 mL/L, FeCl3/6H2O 5x10E-4 g/L, Vitamin B12 5x10E-6 g/L, HCl-Thiamine (B1) 1x10E-4 g/L).
Mass culture: EGG-medium (Glucose 0.3 g/L, Na-acetate 0.3 g/L, (NH4)2SO4 0.1 g/L, MgSO4/7H2O 0.05 g/L, KCl 0.05 g/L, CaCO3 0.05 g/L, 0.2M phosphate buffer solution 5 ml/L, FeCl3/6H2O 5x10E-4 g/L, Vitamin B12 5x10E-6 g/L, HCl-Thiamine (B1) 1x10E-4 g/L) + Agar layer (0.5 g/50 mL), 25 degrees C, static, 7-10 days.
The Type 021N harvest using pipet, and then washing by 1/750 M phosphate buffer at three times. Ultrasonics, 10 degrees C stock.
Reference: T. Kohno; Morphology, physiology, and nutrition of a sulphur-oxidizing filamentous organism isolated from activated sludge, Wat. Sci. Tech.,20, 241-247 (1988).
2.2. Algae (for algal-feeders)
Anabena No.1, No.77 (NIES) Stock culture: MA-medium or M11-medium. 25 degrees C, shaking, 3 weeks, about 2,000 lux.